The seeding of amyloid-β 40 (Aβ40) oligomers from monomers is the initial step of Aβ aggregation, and many reports have suggested that cholesterol enhances this step. We studied the potential of secosteroid vitamin D derivatives for Aβ40 aggregation in vitro. The quartz-crystal microbalance technique demonstrated that vitamin D3 does not show any effect on Aβ40 aggregation while vitamin D2 promoted it and docking simulation but that vitamin D2 has high potential in this regard. Thus, stacking of the Phe19 benzene ring in Aβ40 and the C22-C23 double bond in vitamin D2 may alter the energy of these molecules. Electron microscopy revealed the potential of vitamin D2 to increase Aβ40 aggregation. Thioflavin-T assays indicated that Vitamin D2 induced increased fluorescence at 490 nm, as typically observed for amyloid fibrils but also for protofibrils; in both cases this reflects of the increase of β-sheet contents. Aβ40 aggregation was further confirmed in ELISA, SDS-PAGE and dot blot analysis which revealed changes in protease K resistance. These results suggest a possible mechanism, of how vitamin D2 could increase Aβ40 aggregation and the docking simulation explains, why the same is not observed with vitamin D3.